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Microprojectile-DNA delivery in conifer species: factors affecting assessment of transient gene expression using the β-glucuronidase reporter gene

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Abstract

The Biolistic® microprojectile DNA-delivery method was used to test the usefulness in conifers of eight gene constructs based on the 35S promoter, the AMV translational enhancer, and gene fusion between the P-glucuronidase and the neomycin phosphotransferase II genes. The evaluation was done with embryogenic cells of Picea glauca, where the relative strengths of the promoters were 35S-35S-AMVE>35S-AMVE>35S-35S>35S as evaluated by transient gene expression. The fusion gene of GUS and NPT II gave lower levels of transient gene expression than the unfused GUS gene as detected by X-GLU histochemical assays. Experiments comparing the EM promoter of wheat and the 35S-35S-AMVE promoter (with and without fusion between GUS and NPT II) were done in Picea rubens, P. mariana, P. glauca, and Larix x eurolepis. The unfused gene with the 35S-35S-AMVE promoter gave higher levels of transient gene expression than the fused GUS-NPT II gene. The fluorescent MUG assay was more sensitive than the histochemical X-GLU assay to detect the activity of the β-glucuronidase gene.

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Abbreviations

AMV:

alfalfa mosaic virus

AMVE:

alfalfa mosaic virus translational enhancer

EM:

protein of mature wheat embryo

GUS:

P-glucuronidase gene

MUG:

4-methylumbelliferyl β-D-glucuronide

NPT II:

neomycin phosphotransferase

X-GLU:

5-bromo-4-chloro-3-indolyl β-D-glucuronic acid

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Authors and Affiliations

  1. Molecular Genetics and Tissue Culture Group, Petawawa National Forestry Institute, Forestry Canada, K0J 1J0, Chalk River, Ontario, Canada

    Pierre J. Charest, Nathalie Caléro, Denis Lachance & Luc C. Duchêsne

  2. Plant Biotechnology Institute, National Research Council, 110 Gymnasium Place, S7N 0W9, Saskatoon, Saskatchewan, Canada

    Raju S. S. Datla & Ed W. T. Tsang

Authors
  1. Pierre J. Charest
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  2. Nathalie Caléro
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  3. Denis Lachance
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  4. Raju S. S. Datla
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  5. Luc C. Duchêsne
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  6. Ed W. T. Tsang
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Communicated by F. Constabel

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Charest, P.J., Caléro, N., Lachance, D. et al. Microprojectile-DNA delivery in conifer species: factors affecting assessment of transient gene expression using the β-glucuronidase reporter gene. Plant Cell Reports 12, 189–193 (1993). https://doi.org/10.1007/BF00237051

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  • DOI: https://doi.org/10.1007/BF00237051

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